Division I.6.3 Neutralisation

Cells from the collected supernatant shall be removed by centrifugation (2 000 to 4 000 × g) or membrane filtration (0,45 μm) with a low protein binding membrane and the supernatant shall be diluted 1:100 and 1:10 000 in cell culture medium.

Aliquots of a minimum of two supernatant dilutions shall be mixed and incubated for 60 minutes at 15 °C with equal parts of the following reagents separately:

1. (a)

   serum containing group specific antibody against VHSV at a 1:50 (vol:vol) dilution;

2. (b)

   serum containing group specific antibody against IHNV at a 1:50 (vol:vol) dilution;

3. (c)

   pool of antisera against the indigenous serotypes of IPNV at a 1:50 (vol:vol) dilution;

4. (d)

   medium alone (positive control).

From each virus supernatant-serum mixture, at least two cell cultures shall be inoculated with 50 μl each and then incubated at 15 °C. The development of CPE shall be checked as described in point I.5.4.

VHSV strains and isolates that do not react in neutralisation tests shall be identified by IF or ELISA.

Other neutralisation tests of proven similar efficacy may be used instead.